The present invention concerns the field of bacterial biocontrol. More precisely, the invention relates to the identification of chemicals that promote the growth of bacteria inactivating NAHL.
N-acylhomoserine lactones (NAHL) are essential signals for cell-to-cell communication in numerous bacterial populations and communities. The perception of critical concentration of NAHL by bacterial protein sensors (LuxR family) controls the expression of specific genes. The regulatory pathway that links cell quorum to gene expression via the perception of NAHL signal is termed quorum-sensing (QS) (Fuqua et al., 1994). The functions that are regulated through QS are highly diverse (Whitehead et al., 2001), and involve the virulence of animal and plant pathogens, such as Agrobacterium tumefaciens, Erwinia carotovora, or Pseudomonas aeruginosa. 
Initially discovered in a few bacteria (Dong et al., 2000; Leadbetter and Greenberg 2000), NAHL-degrading enzymatic activities have now been reported in Proteobacteria belonging to the Agrobacterium, Bosea, Commamonas, Delftia, Pseudomonas, Ralstonia, Sphingopyxis, and Variovorax genera (Leadbetter and Greenberg, 2000; Huang et al., 2003; Lin et al., 2003; Uroz et al, 2003; Flagan et al., 2003; Hu et al., 2003; Park et al., 2003; d'Angelo-Picard et al., 2005; Jafra et al., 2006), as well as in Actinobacteria and Firmicutes, such as Arthrobacter, Bacillus, Rhodococcus and Streptomyces genera (Dong et al., 2000; Lee et al., 2002; Uroz et al., 2003; Park et al., 2003; Park et al., 2005; Park et al., 2006). These NAHL-degrading bacteria were recovered from different environments such as soil, rhizosphere and biofilm. Noticeably, an extensive analysis of the diversity of NAHL-degrading and NAHL-producing bacteria in the rhizosphere of Nicotiana tabacum (d'Angelo-Picard et al., 2005) suggests that these two functional communities co-exist in a same environment. Moreover, some bacterial isolates belonging to a same genus (Agrobacterium, Pseudomonas, Sphingopyxis, and Variovorax) may be either NAHL producer or degrader. In a few genera, such as Agrobacterium and Pseudomonas, these two antagonist functions may be co-expressed in a same isolate, and are therefore under a sophisticated regulation, which was explored in the case of Agrobacterium tumefaciens (Zhang et al., 2002; Chevrot et al., 2006).
Over past years, antivirulence strategies targeting QS appeared in the literature (Dong et al., 2001; Zhang 2003; Molina et al., 2003; Rasmussen and Givskov, 2006). They include the following approaches:                inhibition of the synthesis of the NAHL signal: for example, triclosan is an antibiotic molecule which inhibits the synthesis of fatty acids, which are themselves necessary to the synthesis of NAHL (Hoang and Schweizer, 1999). However, resistances to this antibiotic appear because it is not specific for the NAHL.        inhibition of the perception of the NAHL signal: NAHL analogues, such as halogenated furanones, can be used to disturb the NAHL signal recognition by the bacterial receptors (Manefield et al., Microbiology. 1999 February; 145 (Pt 2):283-91).        enzymatic degradation of the NAHL signal by genetically modified organisms: recombinant vectors for the expression, in a microorganism or in a transgenic plant, of an exogenous lactonase or acylase, have been proposed (Dong and Zhang, 2005; US 2004/0139495; US 2005/0155088). Transgenic plants expressing an exogenous lactonase, for example encoded by the aiiA gene from Bacillus sp., are more resistant to an infection by pathogenic bacteria producing NAHL than their unmodified counterparts. However, this approach is not easy to perform, since it necessitates obtaining transgenic plants.        isolation of NAHL-degrading bacteria, for use as biocontrol agents (Uroz et al., 2003; Jafra et al., 2006) such as bacterial strains belonging to the Bacillus, Rhodococcus and Delftia genera.        
In this context, the inventors have investigated whether the metabolic diversity of natural bacterial communities may be engineered to favour NAHL-degrading bacteria through the application of chemicals that promote their growth. As described below, they have demonstrated that NAHL-degrading bacteria may indeed be specifically stimulated, offering a potential strategy for the control of QS-dependent plant pathogens by bacterial population engineering.
According to a first embodiment, the present invention pertains to a soil additive containing at least one compound selected amongst the compounds of formula (I):

wherein R1, R′1, R2 and R3 represent a hydrogen atom, a linear or branched C1-C20 alkyl group, a linear or branched C2-C20 alkylene group carrying one or more double bonds, or a C1-C20 alcoxy group, provided that at least one of R1, R′1, R2 and R3 is an alkyl group, an alkylene group or an alcoxy group.
According to a preferred embodiment of the invention, R1, R′1, R2 or R3 represent a linear or branched C2-C14, preferably C2-C8 and more preferably C2-C6 alkyl group or a linear or branched C2-C14, preferably C2-C8 and more preferably C2-C6 alkylene group carrying one or two double bonds.
In a particular embodiment of the soil additive according to the invention, R1, R′1, R2 or R3 represent an alkyl group or an alkylene group as defined here above, which is substituted with one or more groups selected in the group consisting of hydroxyl, and ketone.
According to another preferred embodiment of the invention, the compound present in the soil additive according to the invention is selected among the following compounds:                compounds in which three of R1, R′1, R2 or R3 are hydrogen atoms and the other one is an alkyl group or an alkylene group; preferably R1 represents an alkyl group of the formula CH3—(CH2)n— with 1≦n≦6.        compounds in which two of R1, R′1, R2 or R3 are hydrogen atoms and each of the other ones is an alkyl group or an alkylene group; preferably R1 represents an alkyl group of formula CH3—(CH2)n— with 1≦n≦6 and R2 represents a methyl group.        compounds in which one of R1, R′1, R2 or R3 is an hydrogen atom and each of the other ones is an alkyl group or an alkylene group; preferably R1 and R′1 represent a methyl group and R2 represents CH3—CO—(CH2)2.        
Preferred examples of soil additive according to the invention comprise at least the following compounds:                gamma-caprolactone (GCL) of formula (II):        
                4-heptanolide (HTN) of formula III:        
                gamma-octalactone (GOL) of formula IV:        
                4-hydroxy-4-methyl-3-(3-oxobutyl)-valeric acid gamma lactone of formula V:        

The soil additive according to the invention preferably contains at least gamma-caprolactone (GCL) or 4-heptanolide (HTN).
In certain situations, the skilled artisan can choose to add, in addition to a compound favouring the growth of NAHL-degrading bacteria, one of several strains of such bacteria. This can be the case, for example, in hydroponic cultures likely to be contaminated by plant pathogens, or in specific soils naturally devoid of NAHL-degrading bacteria. Hence, the present invention also pertains to a soil additive as described above, which further comprises at least one NAHL-degrading bacterial strain, especially a NAHL-degrading bacterial strain, the growth of which is stimulated by gamma-caprolactone (GCL) or 4-heptanolide (HTN). Non-limitative examples of NAHL-degrading bacterial strains that can be used according to this embodiment are bacteria which belong to a genus selected amongst the genera Delftia, Rhodococcus, Ochrobactrum, Pseudomonas, Rhizobium, Sinorhizobium, Bacillus, Comamonas and Variovorax. Preferred strains which can be incorporated to the soil additives of the invention are Delftia acidovorans and Rhodococcus, including the bacterial strain Rhodococcus erythroplis W2 (Uroz et al., 2003). Of course, several strains can be used in combination.
Another aspect of the present invention is the use of a compound of formula (I):

wherein R1, R′1, R2 and R3 represent a hydrogen atom, a linear or branched C1-C20 alkyl group, a linear or branched C2-C20 alkylene group carrying one or more double bonds, or a C1-C20 alcoxy group provided that at least one of R1, R′1, R2 and R3 is an alkyl group, an alkylene group, or an alcoxy group, for favouring the growth of a NAHL-degrading bacterial strain, in a complex bacterial consortium. Of course, the same particular and preferred compounds as described above for the soil additives are also a part of this aspect of the invention.
In particular, these compounds can be used for preventing biofilm formation and/or for preventing the NAHL-dependent expression of a virulence factor from a pathogenic bacterium in a complex environment. Non-limitative examples of such complex environments which can be treated according to the present invention are a soil, a surface likely to be colonized by bacteria, and the interior of a plumbing material, a pipe, a silo, a fermenter, or a colander.
According to another preferred aspect, the present invention pertains to the use of a soil additive as described above, for protecting plants from biofilm formation and/or from the NAHL-dependent expression of a virulence factor from a pathogenic bacterium. This aspect of the invention can be used for protecting plants either in hydroponic cultures, or in soil cultures. Examples of plant pathogens which can be targeted by the invention are: Erwinia sp., especially Erwinia carotovora and Erwinia chrysantemi, Burkholderia sp., especially Burkholderia cepacia, Burkholderia glumae, Burkholderia plantarii, Agrobacterium tumefaciens, Pantoea sterwartii and Ralstonia solanacearum. Accordingly, non-limitative examples of plants which can benefit from the present invention are: potato plants, tomato plants, lettuce, rice, basil, beet.
A process for determining if a compound is able to stimulate NAHL-degrading bacteria in a bacterial consortium containing at least one Delftia strain and/or at least one Rhodococcus strain is also part of the present invention. Such a process can comprise the following steps:
(i) incubating a sample of said bacterial consortium in a synthetic medium enriched with said compound to be tested;
(ii) incubating a sample of said bacterial consortium in the same synthetic medium as in (i), but enriched with one of the compounds of formula II-V and preferably with compound of formula II (GCL) or compound of formula III (HTN) in place of said compound to be tested;
(iii) incubating a sample of said bacterial consortium in the same synthetic medium as in (i), without addition of one of the compounds of formula II-V, nor of the compound to be tested; and
(iv) after an incubation time of 12 h, preferably of at least 20 h, comparing the ability of each of the consortia obtained in the conditions mentioned in (i), (ii) and (iii) to degrade a NAHL compound such as C6-HSL.
When performing this process, the skilled artisan will consider that the tested compound is able to stimulate NAHL-degrading bacteria if the bacterial consortium obtained in step (i) can degrade said NHL compound as least as efficiently as the bacterial consortium obtained in step (ii). Of course, steps (i) to (iii) can be performed simultaneously or sequentially.